Introduction

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Interleukin (IL)-9 is a T-cell-derived cytokine that has been implicated as an important factor in the development of allergic pulmonary inflammation (1, 2). This cytokine belongs to the same cytokine superfamily as IL-4 and shares the IL-2 receptor -chain with IL-4 during signal transduction (3). IL-9 has a variety of effects on inflammatory cells. It stimulates proliferation of primitive hematopoietic pluripotent progenitor cells, enhances immunoglobulin (Ig) E production by B cells, promotes the differentiation and proliferation of mast cells and T cells (1, 2, 4), and induces chemokine expression in lung epithelial cells (8). In transgenic mice that express IL-9 under the control of lung-specific promoter, there is evidence of pulmonary inflammation, increased numbers of mast cells in the airway tissues, elevation in serum IgE, and hypertrophy of airway epithelial cells. Furthermore, airway hyperresponsiveness (AHR) occurs in these mice (9, 10). In humans, the expression of IL-9 messenger RNA (mRNA) is increased in the airways of asthmatics and this increased expression is highly correlated to the decreased FEV₁ and increased airway responsiveness to methacholine (MCh) (11).

The aim of this study was to evaluate the role of IL-9 on inflammation and AHR in allergic mice. Previous studies have identified an important role for T helper (Th) 2-dependent cytokines, including IL-4 and IL-5, on the development of eosinophilic lung inflammation and AHR in this species (12). In this study, a neutralizing monoclonal antibody to murine IL-9 (anti-mIL-9 mAb) was given to allergic mice to evaluate the role of IL-9 on eosinophilic lung inflammation, on changes in lung histopathology, including effects on airway epithelium and goblet cells, serum levels of IgE, steady-state mRNA levels of IL-4, IL-5, IL-13, and interferon (IFN)-, and on the AHR to MCh.

	Materials and Methods

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Sensitization and Antigen Challenge

Young male B6D2F1 mice (Jackson Laboratories, Bar Harbor, ME) were sensitized by intraperitoneal injection of 15 µg ovalbumin (OVA) (Sigma, St. Louis, MO) adsorbed onto 2 mg of aluminum hydroxide gel (alum, Rehydragel; Reheis, Berkeley Heights, NJ) (15). Nonsensitized mice received intraperitoneal injection of alum only. On Day 5, the mice were given an intraperitoneal booster injection of the same antigen or alum. Twelve days after sensitization, the mice were challenged with aerosolized OVA (0.5%) that was generated by an ultrasonic nebulizer (Ultra-Neb 99; DeVilbiss, Somerset, PA). The aerosol was circulated through a large Plexiglas chamber, and mice were placed into the chamber for 1 h in the morning and again in the afternoon (6 h apart) of a single day. Twenty-four hours after the morning OVA aerosol challenge, the mice were prepared for the collection of bronchoalveolar lavage fluid (BALF) and lung tissues, or for the assessment of bronchoconstrictor reactivity to MCh in separate experiments.

Collection and Assessment of BALF

The mice were killed by CO₂ inhalation, and the tracheas were catheterized. Two separate 0.3-ml aliquots of isotonic saline were injected into the tracheal cannula and collected as the BALF sample. Total cell counts were performed by hemocytometry. Slides were prepared on a Shandon cytospin 3 (Shandon, Pittsburgh, PA) at 250 rpm for 10 min, fixed with methanol, and stained with Hema 3 (Fisher Scientific, Pittsburgh, PA). Differential counts on at least 200 cells were done to enumerate the eosinophils (EOs) in each sample (15).

Histopathologic Evaluation of the Lung Tissues

The lungs were perfused in situ by flushing the pulmonary vasculature with buffered saline and fixed in 10% phosphate-buffered formalin. Paraffin sections of the lung tissues were then stained with either hematoxylin and eosin for EO identification or periodic acid-Schiff/alcan blue stain (PAS/AB) for goblet cell identification. Using five random areas, the number of EOs in peribronchial and alveolar regions were counted at ×400 magnification. The number of goblet cells was also enumerated in a unit length of airway. Damage to the airway epithelium and presence of mucus in the airway lumen were semiquantitatively graded with numerical scores from 0 to 3 (0, none; 1, slight; 2, moderate; 3, severe).

Measurement of Bronchoconstrictor Reactivity to Methacholine

Twenty-four hours after the first antigen challenge, mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and prepared with tracheal and jugular venous catheters. The animals were artificially ventilated with a computer-assisted ventilator (Flexivent SAV; Scientific Respiratory Equipment, Montreal, PQ, Canada), using a tidal volume of 0.5 ml and a rate of 130 breaths/min. Airway resistance (Raw) was measured with the same system by the forced oscillation technique (16). Briefly, the ventilator was used to deliver two cycles of a 2-Hz sinusoidal volume waveform to the airway. Airway pressure and delivered volume were measured continuously, and airflow was calculated from the derivative of volume. A two-element series, resistance-elastance model, for the airway was fitted to the flow and pressure data to yield values of Raw (17).

Raw was measured during intravenous injection of increasing doses (0.1 to 1 mg/kg) of MCh (Sigma, St. Louis, MO), and the peak value produced by each injection was recorded. Each MCh dose was given in a volume of 70 µl, approximately 5 min apart. A four-parameter logistic equation was fitted to the dose-response curve (GraphPad Prism, San Diego, CA) and from this the provocative dose of MCh that increased Raw by 100% above baseline (PD₁₀₀) was calculated (17).

RNA Isolation and Semiquantitative Polymerase Chain Reaction

At 6 h after the first OVA challenge, total RNA from lung tissue was prepared for the measurement of IL-4, IL-5, IL-13, and IFN- steady-state mRNA by semiquantitative polymerase chain reaction (PCR) as described previously (13, 18). In brief, perfused lungs were removed and immediately frozen on dry ice. Lung tissues from individual mice were solubilized in guanidinium isothiocyanate, and RNA was purified by cesium chloride ultracentrifugation. RNA was transcribed into complementary DNA (cDNA) and serial dilutions prepared for PCR amplification with murine hypoxanthine phosphoribosyltransferase (mHPRT), IFN-, IL-4, IL-5, or IL-13 primers. The primers and probes used for mHPRT, IL-4, IL-5, and IFN- had been described previously (18). The primers for murine IL-13 (mIL-13) were 5'-TCTTGCTTGCCT TGGTGGTCTCGC-3' (sense) and 5'-GATGGCATTGCAATTG GAGATGTTG-3' (antisense), and the probe was 5'-TCACACAA GACCAGACTCCCCTGTGCAA-3' (19). Levels of steady-state mRNA for each cytokine were quantitated by comparison to standard curves generated from serial dilutions of cDNA from concanavalin A-stimulated BALB/c mice splenocytes, an abundant source of cytokine mRNA. Derived values were normalized by the constitutively expressed gene product HPRT.

Measurement of IgE and IgG₁

Total serum IgE and IgG₁ from blood samples collected 24 h after antigen challenge were determined by enzyme-linked immunosorbent assay according to the procedures outlined in the assay kit (Pharmingen, San Diego, CA). All serum samples were diluted by a factor of 10 before analysis. OVA-specific IgE was measured by coating microwell plates with 100 ng of OVA and blocking with fetal bovine serum. After incubation with serum samples, biotinylated anti-mIgE and streptavidin-horseradish peroxidase conjugate were added. The substrate tetramethalbenzidine and H₂O₂ were then added, and absorbance values were then measured at 450 nm. Values for serum OVA-IgE levels were expressed in arbitrary units based on the mean value of pooled serum from sensitized control mice (20).

Antibody Treatments

Anti-mIL-9 mAb (200 µg/mouse, clone D-9302C12; Pharmingen) was given intraperitoneally at 2 h before sensitization on Day 1 and followed with the same dose on Days 5, 9, and 12. The last dose (on Day 12) was given 2 h before antigen challenge. Control animals received either normal saline or control IgG (purified hamster IgG; Jackson ImmunoResearch Laboratories, West Grove, PA). In one experiment, a single dose (200 µg/mouse) of anti-IL-9 antibody was given 2 h before antigen challenge.

Statistical Analysis

Data are presented as the mean ± standard error of the mean (SEM). Statistically significant effects between the different treatment groups were determined by analysis of variance and Fisher's least protected difference (StatView; Abacus Concepts Inc., Berkeley, CA). A P value less than 0.05 was accepted as statistically significant.

Animal Care and Use

This study was conducted with prior approval from the Animal Care and Use Committee of Schering-Plough Research Institute, which is a facility accredited by the American Association of Animal and Laboratory Animal Care (AAALAC).

	Results

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OVA challenge to sensitized mice increased the number of EOs and total cells appearing in the BALF 24 h after OVA challenge (Figure 1). Treatment of OVA-sensitized and challenged mice with the anti-IL-9 antibody (200 µg/ mouse, four times intraperitoneally) reduced by 70 to 80% the number of EOs and total cells appearing in the BALF (Figure 1). Treatment with the control antibody (200 µg/ mouse, four times intraperitoneally) had no effect on the BAL eosinophilia and the increase in total cells of sensitized mice after OVA challenge. On the other hand, a single dose of the anti-IL-9 antibody (200 µg/mouse, one intraperitoneal injection 2 h before antigen challenge) had no effect on the BAL eosinophilia or total cells of sensitized mice 24 h after OVA challenge.

View larger version (37K): [in this window] [in a new window]   Figure 1.   Effect of anti-mIL-9 antibody on the number of total cells and EOs in the BALF of allergic mice 24 h after OVA challenge. Anti-mIL-9 antibody or control IgG (200 µg/mouse, four times intraperitoneally) was administered 2 h before OVA sensitization and repeated on Days 5, 9, and 12. The last dose was given 2 h before OVA challenge. The single dose of antibody (200 µg/mouse, once intraperitoneally) was given 2 h before OVA challenge. Values represent mean ± SEM (n = 10/group). *P < 0.05 compared with the sensitized, saline-treated group.

OVA challenge to sensitized mice increased the number of EOs in the peribronchial as well as in alveolar regions of the lungs (Table 1). Furthermore, there was moderate damage to the airway epithelium and an increase in the number of PAS⁺/AB⁺ cells in the bronchial epithelium of sensitized mice after OVA challenge. Treatment of sensitized mice with anti-IL-9 antibody (200 µg/mouse, four times intraperitoneally) significantly reduced the number of EOs in the peribronchial and alveolar regions, and attenuated the damage to the airway epithelium and increase in PAS/AB⁺ epithelial cells after OVA challenge (Table 1). Treatment with a single dose of anti-IL-9 antibody or a control antibody had no effect on the appearance of EOs in lung tissue, damage to the airway epithelium, or increase in PAS⁺/AB⁺ epithelial cells.

Steady-state mRNA levels of IL-4, IL-5, and IL-13 in lung tissues of OVA-sensitized and challenged mice were increased by 7-, 14-, and 20-fold, respectively, relative to values in nonsensitized, OVA-challenged controls (Figure 2). Steady-state mRNA level for IFN- was increased by only twofold in sensitized, OVA-challenged mice. Treatment of sensitized mice with the anti-IL-9 antibody (200 µg/mouse, four times intraperitoneally) or control antibody had no effect on the increased steady-state mRNA levels of IL-4, IL-5, IL-13, and IFN-.

View larger version (33K): [in this window] [in a new window]   Figure 2.   Effect of anti-mIL-9 antibody on cytokine steady-state mRNA levels in the lungs of allergic mice. Mice treated with anti-mIL-9 Ab, control antibody, or saline were challenged, and lung tissue was collected 6 h later. Individual tissues from five mice per group were analyzed. IFN-, IL-4, IL-5, and IL-13 steady-state mRNA levels were quantitated by comparison to standard curves generated from serial dilutions of cDNA of concanavalin A-stimulated BALB/c mice splenocytes and expressed in mean ± SEM relative units.

Serum levels of total and OVA-specific IgE were increased dramatically in OVA-sensitized mice relative to values in nonsensitized controls. Treatment with the anti-IL-9 antibody (200 µg/mouse, four times intraperitoneally) reduced serum levels of total and OVA-specific IgE. Treatment with control antibody had no effect (Table 2). Serum level of total IgG₁ was moderately increased in the sensitized mice (1.36 ± 0.17 mg/ml) compared with that of the nonsensitized control mice (0.47 ± 0.15 mg/ml). Treatment with anti-IL-9 Ab (200 µg/mouse, four times intraperitoneally) did not cause significant changes in the serum IgG₁ level (1.45 ± 0.26 mg/ml).

OVA challenge to sensitized mice increased the bronchoconstrictor reactivity to MCh by twofold (PD₁₀₀ = 0.091 ± 0.01) relative to values in OVA-challenged, nonsensitized controls (PD₁₀₀ = 0.18 ± 0.02). Treatment of OVA-sensitized and challenged mice with the anti-IL-9 antibody (200 µg/mouse, four times intraperitoneally) significantly reduced bronchoconstrictor reactivity to MCh (PD₁₀₀ = 0.15 ± 0.01), relative to values in OVA-sensitized and challenged mice treated with control antibody (PD₁₀₀ = 0.095 ± 0.01) (Figure 3). Treatment of sensitized and challenged mice with a single dose of the anti-IL-9 antibody had no effect on the bronchoconstrictor hyperresponsiveness to MCh (PD₁₀₀ = 0.084 ± 0.008, n = 6 per group) (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 3.   Effect of anti-mIL-9 antibody on bronchoconstrictor reactivity to MCh in allergic mice. Anti-mIL-9 antibody or control IgG (200 µg/mouse, four times intraperitoneally) was administered 2 h before OVA sensitization and repeated on Days 5, 9, and 12. The last dose was given 2 h before OVA challenge. Values represent mean ± SEM (n = 6 to 8 per group) of percent increase in airway resistance (Raw). *P < 0.05 compared with the sensitized, saline-treated group.

	Discussion

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In this study, treatment of allergic mice with an antibody to murine IL-9 attenuated the pulmonary eosinophilia and lung damage, the goblet cell hyperplasia, and AHR induced by antigen challenge. The serum levels of total and OVA-specific IgE were also reduced after treatment with the anti-IL-9 antibody, but the serum level of total IgG₁, which is not the major immunoglobulin involved in this particular animal model of allergic inflammation (15), was not reduced after treatment with the antibody. Steady-state mRNA levels of Th2-dependent cytokines, IL-4, IL-5, and IL-13 were unchanged. However, the results in this study are the first to show the inhibitory effect of an anti-IL-9 antibody in a model of murine pulmonary inflammation and AHR induced by antigen challenge.

Data described here are consistent with results from previously published reports using IL-9 transgenic mice (2, 9, 10). Transgenic mice overexpressing IL-9 exhibit enhanced eosinophilic infiltration into the lungs, elevated serum IgE levels, and increased AHR after lung challenge with Aspergillus fumigatus (10). In addition, promoter- directed overexpression of IL-9 in the lungs of transgenic mice resulted in significant EO and lymphocyte infiltration, epithelial cell hypertrophy, and increased AHR (9). In contrast, IL-9-deficient mice have similar serum IgE levels as that of wild-type mice after OVA sensitization (21). Also, these IL-9-deficient mice exhibit normal pulmonary eosinophilia and granuloma formation in response to Schistosoma mansoni infection. It is unclear why the results described in the present report and for the IL-9 transgenic mice are not consistent with those observed with IL-9-deficient mice. Differences in experimental paradigms of antigen sensitization and challenge, differences in the mouse strains, as well as compensatory mechanisms in the IL-9-deficient mice may account for these inconsistent results on the effect of IL-9 on pulmonary inflammation in mice.

The mice used in this study were of the B6D2F1, an F1 hybrid of the DBA2J (D2), which is hyperresponsive to bronchoconstrictor stimuli, and the C57Bl6J (B6) strain, which is hyporesponsive to bronchoconstrictor agents (1, 2). The B6D2F1 is moderately hyperresponsive to bronchoconstrictor challenge under normal conditions, but demonstrates AHR after antigen challenge (2, 17). It is important to note that the inherent airway reactivity in these different strains of mice is positively correlated with the expression of IL-9 in airways (10). The fact that an anti-IL-9 antibody suppressed the development of AHR in B6D2F1 mice after antigen challenge is consistent with the hypothesis that airway reactivity correlates positively with increased expression of IL-9 in the lungs.

Antigen challenge to the sensitized mice increased the presence of EOs in the BALF and lung tissue, produced evidence of lung damage and goblet cell hyperplasia, and generated a condition of AHR. Treatment with the IL-9 antibody effectively inhibited most of this pathology, but it was only effective when it was given during the sensitization period. It had no effect when it was given as a single dose 2 h before the antigen challenge. These results suggest that IL-9 is important during the early stages of the immune response. IL-9 promotes IgE production from B cells (4), and we found that both total and OVA-specific IgE were reduced by treatment with the anti-IL-9 antibody given during the sensitization period. A decrease in IgE production seen in this study would likely suppress the responses to antigen challenge. Indeed, a reduction in the levels of serum level IgE is believed to be one of the major factors behind the anti-inflammatory effects of an antibody to IL-4 (12, 22).

Mast cells are involved in the development of pulmonary eosinophilia in this murine model of allergen-induced lung inflammation (23), although it is important to note that other murine models, particularly those that involve multiple challenges with antigen, are less dependent on mast cell involvement (20, 24). IL-9 has been identified as a mast cell growth factor that enhances the proliferation and upregulation of proteases in mast cells (21, 25, 26). Furthermore, mast cell numbers are increased in the lungs of transgenic mice that overexpressed IL-9 (9). Therefore, an interaction of the anti-IL-9 antibody on mast cells cannot be ruled out.

Pulmonary eosinophilia is a predominant feature of human asthma and is believed to contribute to the pathology of this disease (27). In mice, eosinophilic pulmonary inflammation is induced by sensitization and challenge with OVA, and this effect is triggered, in part, by the action of Th2-dependent cytokines such as IL-4 and IL-5 (12, 22). In this study, treatment with the anti-IL-9 antibody had no effect on the increase in steady-state mRNA levels of IL-4, IL-5, IL-13, and IFN- after antigen challenge. These results are in agreement with the work of Nicolaides and coworkers (1), who showed that IL-4, IL-5, and IFN- steady-state mRNA, and IL-4 and IL-5 protein levels did not correlate with the presence of IL-9 in the lungs of mice. It is important to note that IL-9 enhances the maturation, survival, and upregulation of IL-5 receptor expression on the surface of EOs (28). This raises the possibility that the IL-9 antibody exerts its effect on the interactions of IL-9 with EOs.

IL-9 has also been implicated as an important mediator in the expression, growth, and activation of goblet cells in the airways (21, 29, 30). In the present study, treatment with the IL-9 antibody reduced the increase in PAS/AB⁺ epithelial cells in the airways after antigen challenge. However, the inhibition of goblet cell numbers by the IL-9 antibody was only partial (33% inhibition) even though other indices of inflammatory response such as BAL eosinophilia were inhibited to a much greater extent (85% inhibition). These results suggest that IL-9 contributes to, but may not be, the predominant factor accounting for the goblet cell hyperplasia observed after allergen challenge.

Airway hyperreactivity is a primary pathophysiologic feature of human asthma and occurs in allergic mice after antigen bronchoprovocation. Many factors contribute to AHR, including the presence of lung inflammation, a reduction in basal airway tone and neural mechanism (31). In this study, IL-9 was identified as an important factor involved in the development of airway hyperreactivity after antigen challenge in allergic mice. The studies that were performed with the IL-9 antibody were not designed to elucidate the mechanism for this effect, so it is unknown if IL-9 modulates airway function directly or produces AHR secondary to lung inflammation. However, the allelic variation at the locus for IL-9 correlates with the presence of AHR in murine airways, suggesting that IL-9 is essential for the development of AHR in this species (9).

In conclusion, the results of this study show that an antibody to mIL-9 inhibits the development of allergic pulmonary inflammation and AHR in mice. Regulation of IL-9 expression in the airways may offer a therapeutic approach for the treatment of allergic inflammatory airway disorders.